Studies in Mali on Drug Resistance to Malaria To address the issue of drug resistance in Mali, we have completed a WHO-recommended, therapeutic efficacy study to estimate the effectiveness of artemether-lumefantrine the frontline treatment for uncomplicated P. falciparum malaria in Mali. We found that this treatment is 100% effective in children, and found no evidence for emerging ex-vivo resistance to lumefantrine. Ex-vivo analysis of parasite isolates, however, identified possible decreased sensitivity to quinine. We are currently performing genotype-phenotype analyses to investigate the genetic basis of this finding, and to estimate the current prevalence of other antimalarial drug resistance phenotypes. Malaria Transmission Transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, and we have increased our efforts to investigate parasite sexual stages, malaria transmission in the field, and the biology of the mosquito vector. In May of 2014 we completed a clinical protocol studying 500 individuals of all ages in Kenieroba (13-I-N107). Volunteers were finger-pricked twice per month for a year to analyze both DNA and RNA. We first completed analysis of parasite DNA by PCR directly on filter paper, examining more than 10,000 samples. Using sensitive molecular methods rather than slide reading, we have found that a relatively high proportion of individuals of all ages carry malaria parasites; prevalence was highest in November of 2013 during the wet season and lowest in May of 2014. Unlike most studies, we assessed longitudinal prevalence of parasites in the population and by a linear regression model, only age and gender showed significant effects on the longitudinal prevalence (p<0.0001 and p=0.0008, respectively), while other host factors did not. We showed for the first time that increasing P. falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. More recently we have established the parasite DNA barcoding technique developed at Harvard University to identify different P. falciparum clones. This analysis of 24 different loci in the genome is more sensitive than using polymorphisms in a single gene. In the past year we completed analysis of the numbers of parasite clones in people in the wet and dry seasons. We have found that most individuals in Kenieroba carry more than one clone of P. falciparum and that this distribution does not change from the wet season to the dry season. Finally, we established methods for quantitation of gametocyte mRNA encoding Pfs25 using RT-PCR and applied this technique to analysis of the RNA samples collected in this study. We have determined that 50-80% of people that harbor parasite DNA also have detectable gametocytes throughout the year, showing that no one group can be uniquely targeted for interventions to reduce transmission. Naturally Occurring Wolbachia in Anopheline Mosquitoes from Mali Wolbachia is a bacterium commonly found in various arthropods and is being studied as a vehicle to control insect populations. We have established that Wolbachia infections occur in field-caught adult female Anopheles gambiae s.l. at two localities in Mali. We developed a sensitive qPCR-based detection and quantitation assay for Wolbachia. We found that Wolbachia infected mosquitoes have a lower intensity and prevalence of Plasmodium falciparum sporozoite infection. Wild female mosquitoes were collected in Dangassa and kept in the insectary of the MRTC/ICER Mali for individual oviposition. Both the mothers and the batch of eggs were sent to the Laboratory of LMVR/NIH/NIAID, US, where colonies were established and the presence of Wolbachia was confirmed. We carried out experimental P. falciparum infections, and found that Wolbachia does not affect the early stages of Plasmodium in the mosquito, up to the oocyst stage. However, the prevalence and intensity of sporozoite infection is significantly reduced in females infected with Wolbachia, similar to what was observed in field-collected mosquitoes. Phylogenetic analysis of 16S rRNA sequences indicates that the strain of Wolbachia isolated in A. gambiae mosquitoes from Mali is different from those previously reported in Burkina Faso. Dry Season Ecology of Malaria Vectors We continue to explore the dry season ecology of mosquito vectors and malaria transmission to determine how mosquitoes survive approximately 6 months of the dry season. Several ongoing projects are assessing two different possible mechanisms for this:1) Mosquito dormancy (aestivation) as a strategy of local persistence through the dry season in the Sahel, and 2) Windborne long-range migration of mosquitoes. We have amassed compelling ecological, physiological, and genetic evidence that A. coluzzii (previously, the M form of A. gambiae) persists locally by aestivation whereas A. gambiae (previously, the S form) and A. arabiensis re-colonize Sahelian areas from hundreds of kilometers away after the onset of rains. In our recent study on the genetic variation over time in A. coluzzii, we found additional evidence for aestivation during the dry season as well as long-distance migration in the late rainy season in that species. Ongoing projects include:(i) We have used high altitude aerial (40-250m above ground) sampling of mosquitoes using traps tethered to helium filled balloons. Twenty two A. gambiae s.l. mosquitoes (among thousands of other mosquitoes) were collected at high altitude in the Sahel. Analysis of this windborne migration of malaria vectors is in final stages along with meteorological wind modeling to infer flight trajectories of mosquito migrants. (ii) We have also found Culicine vectors of arboviruses such as Rift Valley Virus in these aerial samples including some new species for Mali;(iii) Study of flight aptitude of wild mosquitoes using tethered flight assays is in final stages of analysis.Monthly assays were performed to test for seasonal variation in flight aptitude among mosquito species. Preliminary analysis showed an expected increase in flight activity correlated temporally with the capture of A. gambiae in aerial samples as well as higher flight aptitude in A. gambiae vs. A. coluzzii. Notably some mosquitoes flew nearly 8 hours during a 9 hour assay; and(iv) A study on seasonal variation in the microbiome of mosquitoes from Sahelian and riparian habitats is near completion.